Anthelmintic resistance in sheep flocks in Ontario, Canada
Abstract
Gastrointestinal
nematodes (GIN) are a significant constraint to pasture-based sheep
production worldwide. Anthelmintic resistance (AR) has been reported in
most sheep-raising areas in the world, yet little is known about the AR
status in Canada. This study was conducted to determine the frequency of
AR in GIN in sheep flocks in Ontario, Canada. Forty-seven sheep flocks
were enrolled in the study, and their level of parasitism was monitored
monthly throughout a grazing season by analyzing owner-acquired fecal
samples from 15 grazing lambs per flock. When the mean GIN fecal egg
count (FEC) reached a threshold of 200 eggs per gram (epg), oral
ivermectin was supplied to producers to check ivermectin efficacy; the
reduction in mean FEC 14 days after ivermectin treatment was calculated.
‘Drench failure’ was defined as a reduction in mean FEC of <95%. In
those flocks with apparent drench failure, researchers performed a Fecal
Egg Count Reduction Test (FECRT), dividing sheep into 4 treatment
groups (n = 10–15): control (i.e. untreated), ivermectin, and,
if sufficient numbers of animals – fenbendazole and levamisole. AR was
defined as a reduction in mean FEC <95% and a lower 95% confidence
interval <90%. Larval cultures were performed on pooled
post-treatment FECRT samples. Larval Development Assays (LDAs) to detect
the presence of resistance to thiabendazole and levamisole were
performed prior to the ivermectin drench check on pooled owner-acquired
fecal samples that reached the 200 epg threshold. Approximately 89%
(42/47) of the farms reached the FEC threshold of 200 epg; 93% (39/42)
of these farms performed an ivermectin drench check, and 88% (34/39) of
these farms had drench failure. The FECRT was performed on 29 of the 34
farms. Resistance to ivermectin, fenbendazole and levamisole was
demonstrated on 97% (28/29), 95% (19/20) and 6% (1/17) of the farms
tested, respectively, with considerable variability in resistance levels
among farms. Haemonchus sp. was the most commonly cultured
parasite from post-treatment fecal samples. LDA results for 21 farms
were available; of these, 14% (3/21) and 62% (13/21) had low and high
levels of thiabendazole resistance, respectively, while none of the
farms exhibited resistance to levamisole. Amongst these tested farms,
resistance to both ivermectin and benzimidazoles was very common. These
findings strongly suggest that AR, particularly in Haemonchus
sp., is a serious problem in these sheep flocks. Thus, marked changes in
GIN management need to be instituted immediately to mitigate a
worsening situation.
Keywords
- Gastro-intestinal nematodes;
- Ivermectin drench failure;
- Fecal Egg Count Reduction Test;
- Anthelmintic resistance;
- Larval Development Assays;
- Post-treatment larval cultures
1. Introduction
Parasitic
gastroenteritis caused by gastrointestinal nematodes (GINs) is widely
considered the most important disease of grazing sheep worldwide,
causing weight loss, diarrhea and death (Sutherland and Scott, 2010).
Gastrointestinal nematode infections are typically controlled with
anthelmintic drugs, and sheep producers worldwide have customarily
relied heavily on such drugs to maintain sheep health and productivity,
while improving the overall profitability of the sheep industry (Sargison, 2008).
In
North America, three broad-spectrum anthelmintic drug classes are most
commonly used in sheep: macrocyclic lactones (e.g. ivermectin and
moxidectin), benzimidazoles (e.g. thiabendazole, fenbendazole and
albendazole) and imidazothiazoles (e.g. levamisole) (Adams, 2001). In Canada, only ivermectin is licensed for use in sheep (Compendium of Veterinary Products, Canada, 2012). Thiabendazole was the first benzimidazole to be marketed in Canada in the early 1960s (Adams, 2001),
but was subsequently replaced with other structurally similar, but
improved drugs, such as fenbendazole and albendazole. Fenbendazole and
albendazole are licensed for use in Canada in cattle (Compendium of Veterinary Products, Canada, 2012),
but are often used in sheep in an extra-label manner. Levamisole has
not been licensed for use in sheep in Canada for the past 10 years (Health Canada – Drug Product Database Online Query, 2012).
Anthelmintic
resistance (AR) is defined as the “heritable ability of the parasite to
tolerate a normally effective dose of the anthelmintic” (Abbott et al., 2009),
and if sufficiently prevalent in a parasite population, results in
treatment failure. However, treatment failure may also be caused by
other confounding factors (McKenna, 1990), such as under-dosing or incorrect administration of anthelmintic drugs (El-Abdellati et al., 2010).
Anthelmintic resistance is an escalating problem in most sheep-rearing countries worldwide (Papadopoulos, 2008), and is a threat to both agricultural income and sheep welfare (Wolstenholme et al., 2004). It is widespread in New Zealand (Waghorn et al., 2006), Australia (Love et al., 1992 and Besier and Love, 2004), and in many South American countries, such as Brazil and Uruguay (Waller et al., 1996 and Cezar et al., 2010). In recent years, AR has also been described in the United States (Kaplan and Vidyashankar, 2012) and in several European countries including Greece (Gallidis et al., 2009), Italy (Cringoli et al., 2009) and the United Kingdom (Jackson and Coop, 2000). In 2007, the first case of AR in Canada was described in a sheep flock in Ontario (Glauser et al., 2007). Ontario is considered to have a humid continental climate, with cold snowy winters and warm to hot summers (World Maps of Köppen-Geiger Climate Classification, 2012). While recent studies have investigated how this climate affects the epidemiology of GIN infections in sheep (Mederos et al., 2010),
no surveys have been published on how widespread the problem of
treatment failure and AR is in sheep flocks in Canada and, in
particular, Ontario.
The Fecal Egg Count Reduction Test (FECRT) is the standard test for determining AR under field conditions (Coles et al., 1992),
and provides an indirect measurement of anthelmintic efficacy by
determining the reduction in fecal egg counts (FECs) after treatment (McKenna, 2006). Several authors have suggested different threshold values for defining the presence of AR (McKenna, 1990, Wood et al., 1995 and Smart, 2009),
but the most commonly accepted threshold is that endorsed by the World
Association for the Advancement of Veterinary Parasitology (WAAVP),
which defines AR as a Fecal Egg Count Reduction (FECR) of <95% and a
lower 95% confidence interval (CI) of <90%; if only one of these two
factors is present, the farm is defined as being ‘suspected’ of having
resistance (Coles et al., 1992).
Despite being the standard test for AR determination, the FECRT is laborious, expensive and time-consuming (Craven et al., 1999 and El-Abdellati et al., 2010). As a result, various alternative diagnostic tests have been suggested for the determination of anthelmintic susceptibility (Coles et al., 2006). The Larval Development Assay (LDA) described by Taylor (1990)
is based on culturing a known number of GIN eggs in the presence of
different anthelmintics. It is reported to be relatively easy to
perform, more sensitive than the FECRT, and allows for the
identification of parasite larvae to the genus level (Taylor, 1990). However, some LDAs are unable to reliably detect resistance to avermectins (Grimshaw et al., 1994),
and the methodology is considered by some to require a high level of
technical expertise, thus limiting its use outside of research
laboratories (Kaplan and Vidyashankar, 2012).
The
objectives of this study in Ontario sheep flocks were: (i) to determine
the frequency of ivermectin treatment failure; (ii) to determine the
frequency of resistance to ivermectin, fenbendazole and levamisole using
a FECRT; and (iii) to assess the frequency of resistance to
thiabendazole and levamisole using the LDA.
2. Materials and methods
2.1. Number and selection of sheep farms
The
study was conducted in Ontario, Canada, for two consecutive grazing
seasons (May to November 2010 and May to November 2011). The target
population was sheep farms in Ontario, while the study population was
eligible and willing sheep producers (as defined below) that were
members of the Ontario Sheep Marketing Agency (OSMA) – a
producer-operated agency formed under the Ontario Farm Products
Marketing Act that represents all producers that raise and sell market
lambs. All registered producers (n = 3600) receive the magazine
‘Ontario Sheep News’, with over 70% of these producers also receiving
emails from the OSMA email list-serve (OSMA office, personal
communication).
Forty-seven
sheep farms were recruited during the summers of 2010 or 2011. This
sample size is associated with a precision of 14%, using a 95% level of
confidence and an estimated ivermectin drench failure prevalence of 60%
(latter estimate based on an unpublished pilot study conducted on
Ontario sheep farms in 2009, in which 8 of 13 farms tested had
ivermectin drench failure).
Recruitment
required volunteer participation and was carried out through talks
given at various OSMA sheep producer meetings held across the province
and letters posted in the ‘Ontario Sheep News’ and distributed via the
email list-serve. To be included in the study, farms had to: (i) have a
minimum of 30 animals (lambs or yearling ewes) in their first grazing
season; and (ii) keep the animals on pasture for at least 3 months
during the grazing season. The first criterion was set to include both
lambs and yearling ewes since a number of producers in Ontario opt to
keep lambs indoors during the summer months to reduce risk of predator
attacks, thereby minimizing their GIN exposure and immunity development
during their first year of life. This study was approved by the Animal
Care Committee (Protocol Number: 09R056) and the Research Ethics Board
(Protocol Number: 09DC005) at the University of Guelph.
2.2. Farm monitoring
Starting
in May each year, all producers enrolled in the study received a kit
for fecal sample collection. This kit consisted of a Styrofoam cooler
with 2 ice-packs, transparent plastic sealable bags, elastic bands,
disposable gloves, cellophane packing tape, a bag with 5 g of coffee
beans (to provide producers with a visual estimate of approximately 5 g
of sheep feces), a courier (Purolator Inc., Canada) shipping label, and a
consent form. Producers were asked to collect 5 g of fresh fecal
pellets from the ground or per rectum, using the gloves provided, from
each of 15 lambs or yearling ewes in their first grazing season, and to
package these fecal samples individually, using the plastic sealable
bags and elastic bands. The producers were then asked to place the
samples in the Styrofoam cooler with the chilled ice-packs, and to
courier the taped box, along with the signed consent form, to the
Parasitology Laboratory, Department of Pathobiology, Ontario Veterinary
College, University of Guelph.
The
fecal samples were analyzed individually and results used to determine
the flock mean FEC, as an indicator of flock-level GIN parasitism. A
mean FEC of 200 eggs per gram (epg) was the threshold used for
conducting the ivermectin drench check, since a previous study has
indicated that this is a sufficient level for detecting changes in FEC
following treatment (Miller et al., 2006).
If the mean FEC on the farm was <200 epg, another kit for fecal
sample collection was sent to the producer, and they were asked to
repeat the process in 3–4 weeks; kits were thus sent approximately
monthly until either the mean FEC attained was ≥200 epg or until early
September. If the mean FEC on the farm reached ≥200 epg, an ivermectin
drench check was conducted.
